engleski

A Proximity Extension Assay (PEA)-based method for quantification of bevacizumab

Introduction: Proximity Extension Assay (PEA) is a direct one-step protein quantification method using a pair of DNA oligonucleotides linked to antibodies against the target molecule. It requires polyclonal or two monoclonal antibodies (mAbs) that bind to target epitopes close enough to form a DNA duplex which is quantified by real-time PCR. Bevacizumab, an anti-cancer drug, is a mAb against vascular endothelial growth factor with common cardiovascular adverse effects. It is widely used off-label to treat neovascular eye disorders by intravitreal application of small doses. Even then, certain amount reaches systemic circulation which is considered relevant regarding safety. We aimed to set-up a PEA-based assay for bevacizumab in human plasma and to preliminary evaluate it in patients treated intravitreally. Methods: We tested (PEA, quantitative PCR) several combinations of commercial mAbs and a Fab fragment against bevacizumab. The best combination was used to quantify bevacizumab in three patients donating plasma before and 24 h after the first intravitreal injection. Results: A combination of a mAb and a Fab fragment (HCA184 and HCA182, Bio-Rad Laboratories, Inc.) performed best: standard curve R-2 0.98, linear dynamic range 1-1000 pM, lower limit of quantification 1 pM (149 pg/mL) and a satisfactory precision (coefficient of variation 12%). All pre-dose patient concentrations were zero, while post-dose concentrations were 10.94, 13.73 and 55.49 ng/mL, in line with previous reports. Discussion: This is the first set-up of a PEA-based assay for quantification of bevacizumab in human plasma. Its good performance and high sensitivity support further evaluation for potential uses particularly when the expected concentrations are low.

Proximity Extension Assay (PEA) ; Bevacizumab ; Anti-VEGF ; Systemic concentration ; Intravitreal administration

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